Ras-like GTP binding proteins comprise an increasingly large family of related proteins, many of which play critical but as yet poorly understood roles in intracellular membrane traffic. In animal cells, members of the rab protein family are of particular interest due to their heterogeneity and characteristic associations with distinct organelles. Several rab proteins have been identified on the endocytic pathway, localizing to either early or late endosomes. Our recent results have demonstrated that the early endosome-associated protein rab4 controls an important regulatory step in the pathway of receptor recycling, possibly in the entry of receptors into vesicles and tubules that are intermediates between early endosomes and the plasma membrane. Since we have also shown that rab4 is a substrate for phosphorylation by p34cdc2 kinase, it may also be at least partly responsible for the arrest of endocytosis in mitotic cells. Experiments described in this proposal are designed to precisely define the function of rab4 on the endocytic pathway and to understand the mechanisms of cell cycle-dependent control of membrane traffic. First, we will evaluate rab4 function by a detailed analysis of the phenotypes associated with the overexpression of wild type and mutant rab4 in transfected cells. These experiments will involve the analysis of intact cells and the development of cell-free assays to reconstitute rab4-dependent functions in vitro. Second, we will compare the function of rab4 with a second early endosome-associated rab protein rab5. This work will involve co-expression of both rab proteins as well as an analysis of rab4/rab5 chimera. Third, to establish the mechanism of rab4 action, we will identify, isolate, and characterize both membrane and cytosolic proteins that interact with rab4. Finally, we will determine whether phosphorylation and dissociation of rab4 from endosomes in mitotic cells is functionally related to the arrest of endocytosis in mitotic cells. Using cells transfected with rab4 mutants that lack the p34cdc kinase phosphorylation site, endocytosis, recycling, and organelle organization will be analyzed using a combination of biochemical assays and real-time imaging of lipid- labeled endocytic organelles by confocal microscopy. These experiments will also provide basic information concerning the biochemical modifications of endosomes in mitotic cells.